Structural Congeners of Izenamides Responsible for Cathepsin D Inhibition: Insights from Synthesis-Derived Elucidation

This study aimed to elucidate the structural congeners of natural izenamides A, B, and C (1–3) responsible for cathepsin D (CTSD) inhibition. Structurally modified izenamides were synthesized and biologically evaluated, and their biologically important core structures were identified. We confirmed that the natural statine (Sta) unit (3S,4S)-γ-amino-β-hydroxy acid is a requisite core structure of izenamides for inhibition of CTSD, which is closely related to the pathophysiological roles in numerous human diseases. Interestingly, the statine-incorporated izenamide C variant (7) and 18-epi-izenamide B variant (8) exhibited more potent CTSD-inhibitory activities than natural izenamides.


Introduction
Marine cyanobacteria are promising natural sources for drug discovery, as they produce diverse secondary metabolites with various biological relevance. Especially, they can produce numerous modified peptides or depsipeptides with various selectivity profiles and therapeutic potential for protease inhibition [1].
Cathepsin D (CTSD) is a lysosomal aspartic protease that plays critical roles in various physiological and pathological processes [2][3][4][5]. It is involved in metabolic proteolysis, energy metabolism, polypeptide hormone and antigen processing, fibrinolysis, activation of enzyme precursors, apoptotic cell death, and maintenance of intracellular homeostasis [2,[6][7][8][9][10][11]. CTSD is widely expressed in various cells of the human body [2,12], with particularly high abundance in the brain. CTSD dysfunction results in the impaired degradation of disease-linked proteins, and is strongly implicated in the pathogenesis of numerous human diseases, especially neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, neuronal ceroid lipofuscinosis, and progressive disorders of the central nervous system [2,4,13,14]. CTSD is also associated with tumor progression, invasion, and metastasis in human malignancies, especially breast cancers, and is therefore a useful biomarker of breast cancer with poor prognosis [2,[14][15][16]. Furthermore, CTSD inhibition can potentially hinder influenza virus replication by modulating host-cell autophagic/apoptotic responses [17]. Given the critical role of CTSD in human diseases, it is considered an attractive molecular target for the treatment of a wide range of diseases. However, novel therapeutic modulators of CTSD have not been sufficiently investigated, highlighting the urgent need for such modulators.
In a previous study, we reported the first total syntheses of izenamides A-C (1)(2)(3) and the structural confirmation of 2. Since then, we have been interested in elucidating the pharmacophore of izenamides through systematic structure investigation. We have been particularly interested in the Sta unit, because izenamides C (3) lacks CTSD inhibition [27] without Sta. We anticipated that the core component responsible for the biological activity of izenamides could be identified through structural variation, followed by biological evaluation. In addition, we desired to develop an optimal izenamide variant structure for enhanced CTSD inhibition. The strategy involved initial studies of the Sta unit, including its stereochemical effects on CTSD inhibition by the most potent izenamide, B (2). The study also involved investigating the Ala units of 1 and 2 and their stereochemical effects on CTSD inhibition. This approach involved the initial preparation of izenamide B variants (4, 5, and 6) consisting of an unnatural diastereomeric Sta unit. A natural Sta-incorporated izenamide C variant (7) was also prepared to confirm the Sta effects of 1 and 2 on CTSD inhibition. To investigate the stereochemical effect of alanine on izenamide B (2), 18-epi-izenamide B (8) consisting of an unnatural D-Ala was prepared ( Figure 1). Here, the design and synthesis of izenamide variants and their biological evaluation as CTSD inhibitors are described. In particular, the core structure of izenamides for CTSD inhibition and the optimized structure for improved CTSD-inhibitory activity were identified. In addition, the  (1)(2)(3) and synthetic izenamide variants (4)(5)(6)(7)(8).
In a previous study, we reported the first total syntheses of izenamides A-C (1-3) and the structural confirmation of 2. Since then, we have been interested in elucidating the pharmacophore of izenamides through systematic structure investigation. We have been particularly interested in the Sta unit, because izenamides C (3) lacks CTSD inhibition [27] without Sta. We anticipated that the core component responsible for the biological activity of izenamides could be identified through structural variation, followed by biological evaluation. In addition, we desired to develop an optimal izenamide variant structure for enhanced CTSD inhibition. The strategy involved initial studies of the Sta unit, including its stereochemical effects on CTSD inhibition by the most potent izenamide, B (2). The study also involved investigating the Ala units of 1 and 2 and their stereochemical effects on CTSD inhibition.
This approach involved the initial preparation of izenamide B variants (4, 5, and 6) consisting of an unnatural diastereomeric Sta unit. A natural Sta-incorporated izenamide C variant (7) was also prepared to confirm the Sta effects of 1 and 2 on CTSD inhibition. To investigate the stereochemical effect of alanine on izenamide B (2), 18-epi-izenamide B (8) consisting of an unnatural D-Ala was prepared ( Figure 1). Here, the design and synthesis of izenamide variants and their biological evaluation as CTSD inhibitors are described. In particular, the core structure of izenamides for CTSD inhibition and the optimized structure for improved CTSD-inhibitory activity were identified. In addition, the efficient synthesis of izenamide variants with minimized epimerization of the stereocenters was explored by employing a versatile and diverse synthetic route to prevent 2,5-diketopiperazine (DKP) formation.

Completion of Izenamide Variant Synthesis
To investigate the role of the stereochemistry of the Ala unit of 2 in CTSD inhibition, we synthesized Sta-incorporated izenamide C (7) and 18-epi-izenamide B (8). The incorporation of a natural Sta unit into izenamide C (3), which contains a Gly unit instead of an Ala unit, was achieved at the corresponding Sta position in natural izenamide B (2). Additionally, the preparation of 18-epi-izenamide B (8) involved the substitution of unnatural D-Ala unit. Following the preparation of intermediate (3R,4S)-11, we focused on the synthesizing unnatural Sta precursors (3R,4R)-12 and (3S,4R)-13, which serve as key fragments for izenamide B variants (5) and (6). Commercially available aldehyde 18 was reacted with the Grignard reagent [29], which led to the nucleophilic addition of allylMgBr to N-Boc-Dleucinal 18 at 0 • C. Diastereomeric mixtures of alcohols 25 and 26 were obtained in 43% and 42% yield, respectively. The structure and stereochemistry of both alcohol 25 and 26 were confirmed by comparison of their spectral data with those of the corresponding enantiomers, including the opposite optical rotation [30]. Acetonide protection of optically pure allylic alcohols 25 and 26 followed by the Ru-mediated oxidation of the terminal alkenes afforded the desired diastereomeric Sta precursors (3R,4R)-12 and (3S,4R)-13, respectively (Scheme 3). Following the preparation of intermediate (3R,4S)-11, we focused on the synthesizing unnatural Sta precursors (3R,4R)-12 and (3S,4R)-13, which serve as key fragments for izenamide B variants (5) and (6). Commercially available aldehyde 18 was reacted with the Grignard reagent [29], which led to the nucleophilic addition of allylMgBr to N-Boc-Dleucinal 18 at 0 °C. Diastereomeric mixtures of alcohols 25 and 26 were obtained in 43% and 42% yield, respectively. The structure and stereochemistry of both alcohol 25 and 26 were confirmed by comparison of their spectral data with those of the corresponding enantiomers, including the opposite optical rotation [30]. Acetonide protection of optically pure allylic alcohols 25 and 26 followed by the Ru-mediated oxidation of the terminal alkenes afforded the desired diastereomeric Sta precursors (3R,4R)-12 and (3S,4R)-13, respectively (Scheme 3).
To investigate the role of the stereochemistry of the Ala unit of 2 in CTSD inhibition, we synthesized Sta-incorporated izenamide C (7) and 18-epi-izenamide B (8). The incorporation of a natural Sta unit into izenamide C (3), which contains a Gly unit instead of an Ala unit, was achieved at the corresponding Sta position in natural izenamide B (2). Additionally, the preparation of 18-epi-izenamide B (8) involved the substitution of unnatural D-Ala unit.
Tripeptide 20 was synthesized by coupling the known acid 32 [27] with N-Me-D-Phe-OMe·TFA salt [27]. The ester hydrolysis of tripeptide 20 and subsequent amidation of the resulting acid with 22 afforded tetrapeptide 15 in a yield of 76% over two steps (Scheme 5).

Scheme 5. Preparation of tetrapeptide 15.
Tetrapeptide 14, acid 9, and natural Sta precursor 10 were prepared following a previously reported synthetic protocol [27]. The synthesis of izenamide variants 7 and 8 (Scheme 6) was completed by Boc-deprotection of tetrapeptides 14 and 15 and subsequent EDC-mediated amide coupling with acid 10. Pentamer intermediates 33 and 34 were obtained in yields of 81% and 76%, respectively, in two steps. The global deprotection of pentamers 33 and 34 and subsequent amide coupling of the resulting amines with acid 9 produced the corresponding heptamers, which were subjected to desilylation to afford Sta-incorporated izenamide C (7) and 18-epi-izenamide B (8) in yields of 62% and 63% over three steps, respectively (All spectral data including 1 H, 13 C NMR and HRMS of izenamides (4)(5)(6)(7)(8) was in Supplementary Materials). To investigate the role of the stereochemistry of the Ala unit of 2 in CTSD inhibition, we synthesized Sta-incorporated izenamide C (7) and 18-epi-izenamide B (8). The incorporation of a natural Sta unit into izenamide C (3), which contains a Gly unit instead of an Ala unit, was achieved at the corresponding Sta position in natural izenamide B (2). Additionally, the preparation of 18-epi-izenamide B (8) involved the substitution of unnatural D-Ala unit.
Tripeptide 20 was synthesized by coupling the known acid 32 [27] with N-Me-D-Phe-OMe·TFA salt [27]. The ester hydrolysis of tripeptide 20 and subsequent amidation of the resulting acid with 22 afforded tetrapeptide 15 in a yield of 76% over two steps (Scheme 5). Tripeptide 20 was synthesized by coupling the known acid 32 [27] with N-Me-D-Phe-OMe·TFA salt [27]. The ester hydrolysis of tripeptide 20 and subsequent amidation of the resulting acid with 22 afforded tetrapeptide 15 in a yield of 76% over two steps (Scheme 5).

Scheme 5. Preparation of tetrapeptide 15.
Tetrapeptide 14, acid 9, and natural Sta precursor 10 were prepared following a previously reported synthetic protocol [27]. The synthesis of izenamide variants 7 and 8 (Scheme 6) was completed by Boc-deprotection of tetrapeptides 14 and 15 and subsequent EDC-mediated amide coupling with acid 10. Pentamer intermediates 33 and 34 were obtained in yields of 81% and 76%, respectively, in two steps. The global deprotection of pentamers 33 and 34 and subsequent amide coupling of the resulting amines with acid 9 produced the corresponding heptamers, which were subjected to desilylation to afford Sta-incorporated izenamide C (7) and 18-epi-izenamide B (8) in yields of 62% and 63% over three steps, respectively (All spectral data including 1 H, 13 C NMR and HRMS of izenamides (4)(5)(6)(7)(8) was in Supplementary Materials).
The natural Sta units of izenamides are essential for binding to CTSD [18]. Co-crystal structural analysis and docking studies predicted that the hydroxy group of the natural Sta in pepstatin A or izenamide A interacts with Asp residues at the active site of CTSD [31]. However, the effects of the stereochemistry of the two stereocenters in the Sta unit on CTSD inhibition remains unexplored. Thus, the stereochemical effects of the Sta unit of the izenamides on their CTSD-inhibitory activity were examined. As shown in Table 1, izenamide B (2) exhibits moderate inhibitory effects against CTSD (8.12% at 0.5 μM and 14.76% at 1.0 μM), whereas the unnatural Sta-incorporated izenamide B variants (4-6) exhibit no CTSD-inhibitory activities. These results confirm that natural (3S,4S)-Sta is essential for CTSD by izenamides.
Notably, the izenamide C variant (7) exhibits more potent CTSD-inhibitory activities (26.99% at 0.5 μM and 36.07% at 1.0 μM) than those of the natural izenamide B (2). The Sta-incorporated izenamide C variant (7) possesses the same peptide sequence as the biologically active 1 and 2, with the exception of alanine being replaced by glycine. The improved CTSD inhibition of the izenamide C variant (7), in contrast to the moderate inhibitory activity of the natural Sta-containing izenamide B (2) against CTSD, indicates that glycine is more appropriate for achieving adequate CTSD-inhibitory activity. These results confirm the crucial role played by the natural Sta unit as a key pharmacophore in the inhibition of CTSD by izenamides.
The sole difference in structure between 2 and 7 was the absence of a methyl substituent on the Gly unit at C18 in the izenamide C variant (7). Therefore, the optimal stereochemistry of the Ala unit in izenamide B (2) for CTSD inhibition was investigated.  was prepared, as shown in Scheme 6. Surprisingly, the 18-epi-izenamide B (8) showed the best CTSD-inhibitory effects (40.96% at 1.0 μM) compared to those of 2 and 7 (14.76% and 36.07% at 1.0 μM, respectively).

CTSD-Inhibitory Activities of Izenamides (1-3) and Their Variants
The natural Sta units of izenamides are essential for binding to CTSD [18]. Co-crystal structural analysis and docking studies predicted that the hydroxy group of the natural Sta in pepstatin A or izenamide A interacts with Asp residues at the active site of CTSD [31]. However, the effects of the stereochemistry of the two stereocenters in the Sta unit on CTSD inhibition remains unexplored. Thus, the stereochemical effects of the Sta unit of the izenamides on their CTSD-inhibitory activity were examined. As shown in Table 1, izenamide B (2) exhibits moderate inhibitory effects against CTSD (8.12% at 0.5 µM and 14.76% at 1.0 µM), whereas the unnatural Sta-incorporated izenamide B variants (4)(5)(6) exhibit no CTSD-inhibitory activities. These results confirm that natural (3S,4S)-Sta is essential for CTSD by izenamides.
Notably, the izenamide C variant (7) exhibits more potent CTSD-inhibitory activities (26.99% at 0.5 µM and 36.07% at 1.0 µM) than those of the natural izenamide B (2). The Sta-incorporated izenamide C variant (7) possesses the same peptide sequence as the biologically active 1 and 2, with the exception of alanine being replaced by glycine. The improved CTSD inhibition of the izenamide C variant (7), in contrast to the moderate inhibitory activity of the natural Sta-containing izenamide B (2) against CTSD, indicates that glycine is more appropriate for achieving adequate CTSD-inhibitory activity. These results confirm the crucial role played by the natural Sta unit as a key pharmacophore in the inhibition of CTSD by izenamides.
The sole difference in structure between 2 and 7 was the absence of a methyl substituent on the Gly unit at C18 in the izenamide C variant (7). Therefore, the optimal stereochemistry of the Ala unit in izenamide B (2) for CTSD inhibition was investigated. The 18-epiizenamide B (8) was prepared, as shown in Scheme 6. Surprisingly, the 18-epi-izenamide B (8) showed the best CTSD-inhibitory effects (40.96% at 1.0 µM) compared to those of 2 and 7 (14.76% and 36.07% at 1.0 µM, respectively). Overall, this study elucidated the crucial role of the natural Sta unit of izenamides in CTSD inhibition, with the importance of its stereochemistry. The results also suggested that the stereochemistry of the natural Sta unit is critical for its appropriate conformation to interact with Asp residues in the active binding pocket of CTSD, as described previously [18,29]. Additionally, the improved CTSD inhibition observed with the epimeric C18 unit of 8 suggested that it enhances interaction with CTSD. Overall, this study elucidated the crucial role of the natural Sta unit of izenamides in CTSD inhibition, with the importance of its stereochemistry. The results also suggested that the stereochemistry of the natural Sta unit is critical for its appropriate conformation to interact with Asp residues in the active binding pocket of CTSD, as described previously [18,29]. Additionally, the improved CTSD inhibition observed with the epimeric C18 unit of 8 suggested that it enhances interaction with CTSD. Overall, this study elucidated the crucial role of the natural Sta unit of izenamides in CTSD inhibition, with the importance of its stereochemistry. The results also suggested that the stereochemistry of the natural Sta unit is critical for its appropriate conformation to interact with Asp residues in the active binding pocket of CTSD, as described previously [18,29]. Additionally, the improved CTSD inhibition observed with the epimeric C18 unit of 8 suggested that it enhances interaction with CTSD. Overall, this study elucidated the crucial role of the natural Sta unit of izenamides in CTSD inhibition, with the importance of its stereochemistry. The results also suggested that the stereochemistry of the natural Sta unit is critical for its appropriate conformation to interact with Asp residues in the active binding pocket of CTSD, as described previously [18,29]. Additionally, the improved CTSD inhibition observed with the epimeric C18 unit of 8 suggested that it enhances interaction with CTSD. (4)(5)(6)(7)(8). Overall, this study elucidated the crucial role of the natural Sta unit of izenamides in CTSD inhibition, with the importance of its stereochemistry. The results also suggested that the stereochemistry of the natural Sta unit is critical for its appropriate conformation to interact with Asp residues in the active binding pocket of CTSD, as described previously [18,29]. Additionally, the improved CTSD inhibition observed with the epimeric C18 unit of 8 suggested that it enhances interaction with CTSD. (4)(5)(6)(7)(8). Sta-incorporated izenamide C (7) 26 Overall, this study elucidated the crucial role of the natural Sta unit of izenamides in CTSD inhibition, with the importance of its stereochemistry. The results also suggested that the stereochemistry of the natural Sta unit is critical for its appropriate conformation to interact with Asp residues in the active binding pocket of CTSD, as described previously [18,29]. Additionally, the improved CTSD inhibition observed with the epimeric C18 unit of 8 suggested that it enhances interaction with CTSD. (4)(5)(6)(7)(8). Sta-incorporated izenamide C (7) 26 Overall, this study elucidated the crucial role of the natural Sta unit of izenamides in CTSD inhibition, with the importance of its stereochemistry. The results also suggested that the stereochemistry of the natural Sta unit is critical for its appropriate conformation to interact with Asp residues in the active binding pocket of CTSD, as described previously [18,29]. Additionally, the improved CTSD inhibition observed with the epimeric C18 unit of 8 suggested that it enhances interaction with CTSD. (4)(5)(6)(7)(8). Sta-incorporated izenamide C (7) 26 Overall, this study elucidated the crucial role of the natural Sta unit of izenamides in CTSD inhibition, with the importance of its stereochemistry. The results also suggested that the stereochemistry of the natural Sta unit is critical for its appropriate conformation to interact with Asp residues in the active binding pocket of CTSD, as described previously [18,29]. Additionally, the improved CTSD inhibition observed with the epimeric C18 unit of 8 suggested that it enhances interaction with CTSD. Table 1. Cathepsin D inhibitory activities of synthetic izenamides A-C (1-3) and their variants (4)(5)(6)(7)(8). Overall, this study elucidated the crucial role of the natural Sta unit of izenamides in CTSD inhibition, with the importance of its stereochemistry. The results also suggested that the stereochemistry of the natural Sta unit is critical for its appropriate conformation to interact with Asp residues in the active binding pocket of CTSD, as described previously [18,29]. Additionally, the improved CTSD inhibition observed with the epimeric C18 unit of 8 suggested that it enhances interaction with CTSD.

General Information
Unless noted otherwise, all starting materials and reagents were obtained from commercial suppliers and were used without further purification. Tetrahydrofuran was distilled from sodium benzophenone ketyl. Dichloromethane, chloroform and acetonitrile were freshly distilled from calcium hydride. All solvents used for routine isolation of products and chromatography were reagent grade and glass-distilled. Reaction flasks were dried at 100 • C. Air-and moisture-sensitive reactions were performed under argon atmosphere. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck, Darmstadt, Germany) with the indicated solvents. Thin-layer chromatography was performed using 0.25 mm silica gel plates (Merck, Darmstadt, Germany). High-resolution mass spectra (HRMS) were recorded by JMS-700 (JEOL, Tokyo, Japan) and Q-TOF 6530 MS (Agilent, Santa Clara, CA, USA). Optical rotations were measured with JASCO P-2000 digital polarimeter (JASCO, Easton, MD, USA) at ambient temperature using a 10 mm cylindrical cell. Infrared spectra were recorded on a JASCO FT-IR-4200 spectrometer (JASCO, Easton, MD, USA). 1 H and 13 C NMR spectra were recorded using BRUKER AVANCE-400 (Bruker, Billerica, MA, USA) and BRUKER AVANCE-800 (Bruker, Billerica, MA, USA). Chemical shifts are expressed in parts per million (ppm, δ) downfield from tetramethylsilane and are referenced to the deuterated solvent (CHCl 3 or MeOH). 1 H NMR data were reported in the order of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; dt, doublet of triplet; dq, doublet of quartet; dqu, doublet of quintet; ddd, doublet of doublet of doublet; qd, quartet of doublet, m, multiplet and/or multiple resonances), coupling constant in hertz (Hz) and number of protons.

Conclusions
In this study, a range of structural variants of izenamide depsipeptides were designed and synthesized to elucidate core structures responsible for CTSD inhibition. The divergent syntheses of four diastereomers of the Sta unit of izenamide B and evaluation of CTSD inhibition by the izenamide variants were conducted. The results confirmed the essential role of the natural (3S,4S)-Sta unit of izenamides A (1) and B (2) in CTSD inhibition. The incorporation of the natural Sta unit into izenamide C (3) led us to identify an izenamide C variant (7) that exhibited more potent CTSD inhibition than izenamide B (2). Moreover, the most potent 18-epi-izenamide B (8), possessing an unnatural D-Ala in izenamide B (2), was identified based on the substituent effects of the alanine unit. These results highlighted the important role of the Ala or Gly unit, in addition to the natural Sta unit of izenamides, in CTSD inhibition. Overall, these findings provide valuable information for the further development of novel depsipeptide-based CTSD inhibitors.